During chronic liver injury, hepatic stellate cells (HSC) produce and deposit excessive quantities of fibrillar collagens leading to scar formation and compromised liver function. This fibrotic process is driven by connective tissue growth factor (CTGF). Our broad long-term objective is to inhibit the production or action or CTGF so that effective anti-fibrotic strategies can be developed for use in humans. The overall objective of this application is to determine the function of microRNA-199a (miR-199a) or mircoRNA-214 (miR-214) in regulating CTGF production in light of our identification of miR-199a/214 as hitherto unrecognized miRs in HSC which function as negative regulators of CTGF mRNA expression. Thus miR-199a/214 are expressed at high levels in quiescent HSC in normal liver thereby inhibiting CTGF production whereas their expression is suppressed during HSC activation after injury leading to enhanced CTGF expression. MiR-199a/214 are exported from HSC via nano-size exosomes, a novel finding that is important since it allows for miR delivery to neighboring cells and regulation of target genes therein. Our central hypothesis is that expression or action of miR-199a/214 in HSC are critical determinants of CTGF mRNA expression. The Aims to test our hypothesis are: [1] Establish mechanisms regulating miR-199a/214 production in HSC [2] Establish the role of miR-199a/214 in HSC activation and fibrosis [3] Establish the role of exosomal miR-199a/214 in intercellular signaling The expected outcome of these studies will be a more complete understanding of the manner in which CTGF production is regulated during HSC activation. The rationale that underlies the proposed research is that once the roles of miR-199a/214 in CTGF regulation are better understood, they may be exploitable as novel anti- fibrotics. Development of new therapies is critical because fibrotic pathology represents one of the largest groups of disorders for which there is no effective therapy. The medical and financial burdens of liver fibrosis are huge: liver cirrhosis is the ninth leading cause of death in the West, affects millions of individuals world- wide, and is a harbinger of hepatic cancer. The proposed studies are responsive to the 2004 trans-NIH Action Plan for Liver Disease Research and will have a positive impact on improving public health by providing new leads in our understanding of CTGF biology, which is central to the process of liver fibrosis and the development of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Fibrosis of internal organs is a significant contributing factor in about 45% of the deaths in the USA yet there are currently no FDA-approved anti-fibrotic therapeutics. Chronic fibrosis most commonly affects the liver, lung, kidney, heart, pancreas and skin and is the result of diverse causes such as autoimmune, genetic, idiopathic, or toxins (e.g. alcohol in hepatic fibrosis). These studies will determine the role of connective tissue growth factor (CTGF) in controlling the pro-fibrogenic functions of hepatic stellate cells, which are the principal fibrogenic cell type in the liver. This proposal focuses on identifying the mechanisms by which CTGF production is regulated by small naturally occurring RNA molecules called microRNA. The relevance of these studies is that they will identify key steps in the pathways that lead to hepatic fibrosis and will define points of therapeutic intervention that exploit the central role played by connective tissue growth factor (CTGF) in driving fibrotic pathology.